Composition comprising midkine or pleiotrophin protein and method of increasing hematopoietic cells

ABSTRACT

The present invention provides novel use of the MK family that is used alone as an agent for proliferating hematopoietic stem cells and hematopoietic precursor cells. The invention also provides an agent for remarkably enhancing the above-described effect for promoting the proliferation of hematopoietic stem cells and hematopoietic precursor cells, comprising the MK family in combination with known hematopoietic factors such as IL-3, IL-6, G-CSF, GM-CSF, M-CSF, SCF, and EPO.

TECHNICAL FIELD

[0001] This invention relates to novel use of MK to promoteproliferation and differentiation of hematopoietic stem cells andhematopoietic precursor cells in hematopoietic tissues, peripheralblood, or umbilical cord blood synergistically with other hematopoieticfactors.

BACKGROUND ART

[0002] In blood, there exist various hemocytes having different shapesand functions, including erythrocytes, leukocytes, and platelets, whichplay important roles in maintaining homeostasis of the living body.These matured hemocytes have their own life-spans. For maintaining thehemocyte count at the constant level, hemocytes must be incessantlyproduced to make up for the number of hemocytes that is lost due to theexpiration of their life-spans.

[0003] In the normal healthy individual, it is presumed that dailyproduction of hemocytes reaches as much as 2×10¹¹ erythrocytes, 10¹¹leukocytes, and 1 to 2×10¹¹ platelets. Hematopoietic stem cells playcentral roles in the system to produce such an enormous number ofhemocytes over a long period without being exhausted. The cells have notonly self-renewal capability but also multipotentiality to differentiateto various mature hemocytes including erythrocytes, granulocytes,platelets, and lymphocytes. Hematopoietic stem cells (multipotentialstem cells) lose their self-renewal capability as they proliferate tobecome hematopoietic precursor cells (committed stem cells) destined todifferentiate to the specific hemocytes. Hematopoietic precursor cellsthen differentiate to matured peripheral hemocytes.

[0004] It has been known that a number of cytokines regulate each stepof the hematopoietic system to proliferate and differentiatehematopoietic stem cells to various mature hemocytes via hematopoieticprecursor cells. At least twenty kinds of these cytokines participatingin the hematopoietic system have been found at present (Masami Bessho:Igaku no Ayumi 180(13): 802-806, 1997). Their genes have been allcloned, allowing their production on a large scale by geneticengineering techniques. Stem cell factor (SCF) and flk-2 ligand are themost remarkable cytokines as factors acting on mainly hematopoietic stemcells at the early stage of hematopoiesis. SCF acts on the mostundifferentiated hematopoietic stem cells. In either mice or humans, itremarkably promotes the formation of colonies of blast colony-formingunit (CFU-BL), colony-forming unit-mixed (CFU-Mix), burst formingunit-erythrocyte (BFU-e), colony-forming unit-granulocyte/macrophage(CFU-GM), eosinophil colony-forming unit (CFU-Eo), and colony-formingunit-megakaryocyte (CFU-Meg), showing a synergistic effect with variouscytokines such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-11, G-CSF,GM-CSF, and EPO. It has been reported that SCF alone has a weakcolony-stimulating activity (Tsuji, K. et al., Blood 78: 1223, 1991;Shioharu, M. et al., Blood 81: 1453, 1993; Kubo, T. and Nakahata, T.,Int. Hematol. 58: 153, 1993). Nevertheless, SCF is thought to be themost important cytokine for in vitro amplification of hematopoietic stemcells at present.

[0005] The gene for flk-2 ligand has been just recently cloned and itsbiological activity has not been fully clarified. Since it exhibitssynergistic actions with many cytokines as SCF does, it is expected tobe an important factor for in vitro amplification of human hematopoieticstem cells.

[0006] Some of these hematopoietic factors have been clinically applied.For example, erythropoietin (EPO), which promotes to produceerythrocytes, is used for treating renal anemia, and granulocytecolony-stimulating factor (G-CSF), which promotes to produce neutrophilsis used for treating neutropenia caused by the cancer chemotherapy.These contribute to improve quality of life of patients. Recently,clinical application of thrombopoietin (TPO) for treatingthrombocytopenia has been studied because it promotes the production ofplatelets.

[0007] On the one hand, since hematopoietic stem cells are capable ofreconstituting all kinds of cells in the hematopoietic system, thetransplantation of hematopoietic stem cells has been widely performedfor hematopoietic tumors. Recently, the transplantation of peripheralblood stem cells has become rapidly prevalent, and gathered attention asthe powerful fundamental therapy for the chemotherapy-sensitivemalignant tumors including the hematopoietic organ tumors. Furthermore,as a future prospect, the transplantation of hematopoietic stem cells isexpected to be introduced to many of the cell therapy and gene therapy.For that purpose, it is necessary to establish a method for amplifyinghematopoietic stem cells in vitro. However, even now, humanhematopoietic stem cells have been neither isolated nor clarified as towhat extent they can repeat self-renewal.

DISCLOSURE OF THE INVENTION

[0008] An objective of the present invention is to provide a novelcytokine capable of synergistically promoting the proliferation anddifferentiation of hematopoietic stem cells or hematopoietic precursorcells in combination with known cytokines.

[0009] Another objective of the present invention is to provide a novelcytokine capable of promoting the proliferation and differentiation ofgranulocyte/monocyte precursor cells.

[0010] Still another objective of the present invention is to provide anovel cytokine capable of promoting the proliferation anddifferentiation of erythroblast precursor cells in combination withknown cytokines.

[0011] The present inventors have found that single use of a novelgrowth factor called midkine (MK) or pleiotrophin (PTN) promotes theproliferation and differentiation of hematopoietic stem cells andhematopoietic precursor cells (also called hematopoietic cells) ofmammals such as mice and humans in vitro. MK also exerts an extremelyremarkable synergistic effect for proliferating and differentiatinghematopoietic cells when used together with SCF, M-CSF, G-CSF, GM-CSF,IL-3, and IL-6. Furthermore, the inventors have found that MK or PTNpromotes rapid recovery of neutrophils in neutropenia of mammals.

[0012] The present invention will be described in detail below.

[0013] MK was isolated as the product of a gene that is expressed at theearly stage of the differentiation of mouse embryonic tumor cells by theinduction of retinoic acid (Kadomatsu, K., et al., Biochem. Biophy. Res.Commun. 115: 1312-1318, 1988). PTN was found as a heparin-bindingprotein with the neurite outgrowth capability in the newborn rat brain(Rauvaa, H., EMBO J. 8: 2933-2941, 1989). MK and PTN belong to a newclass of haparin-binding growth factors, sharing a 45% homology (inamino acid sequence) each other, and called as the MK family. MK and PTNrespectively exhibit characteristic expression patterns in thedevelopmental process, indicating that they have important physiologicalactivities for the implementation of differentiation.

[0014] Paying attention to such biological activities of the MK family,the present inventors studied their hematopoietic factor activities toproliferate and differentiate myeloid cells and peripheral blood stemcells of mammals. In general, in what stage of theproliferation/differentiation process of hematopoietic stem cells andhematopoietic precursor cells in myeloid hematopoietic factorsparticipate and function can be studied by culturing a certain number ofmyelocytes in a semi-solid medium in the presence of these hematopoieticfactors, selecting cells constituting colonies formed, and counting thenumber of colonies. In such colony formation methods, it has been provedby a number of direct or indirect methods that, a single hematopoieticprecursor cell proliferates, divides, and matures, forming a singlecolony comprising many matured hemocytes. There are colony formationassay methods specific for cells of each hematopoietic system includinggranulocyte/macrophage, erythroblasts, and megakaryocytes, andstimulators specific for each hematopoietic system are used.

[0015] Precursor cells of the granulocyte/macrophage system, CFU-GM,differentiate to precursor cells of neutrophil system, CFU-G, andprecursor cells of monocytes, CFU-M. For that purpose, colony-simulatingfactors (CSF) specific to each precursor cell must be present. Morespecifically, GM-CFS is required for CFU-GM, G-CSF for CFU-G, and M-CSFfor CFU-M. Some of these CSFs not only differentiate precursor cells tomature cells but also activate the function of matured hemocytes.

[0016] CFU-GM can be cultivated by either the soft agar method or themethylcellulose method using bone marrow nucleated cells. Since coloniesformed by either method are constituted by a cell population ofgranulocytes and macrophages at various developmental stages, precursorcells one step further differentiated from hematopoietic stem cells areto be examined. Picking up and staining of these colonies revealed thepresence of G colony consisting of granulocytes, M colony consisting ofmacrophages, and GM colony consisting of the mixture of both. In humans,colonies are rather small and classified into a group called colonycontaining 40 cells or more and a group called cluster with loweraccumulation of cells than colony.

[0017] In order to examine whether MK has the activity to proliferatemyelocytes, the proliferation of mouse myelocytes was assayed by the MTTmethod, resulting in enhancing the proliferation 1.6- to 2-fold in thesystem supplemented with MK at the concentrations of 5, 50, 500, and5000 ng/ml as compared with the system without MK. Aconcentration-dependent elevation of the activity was observed in therange of 5 to 500 ng/ml MK.

[0018] In the colony assay for human peripheral blood mononuclear cellsin the presence of various cytokines, as shown in FIG. 1, colonies werenot formed at all in the system without cytokines, but formed in thesystem supplemented with MK similarly as with GM-CSF and IL-3. Thecolony size tends to be larger in the system added with MK than in thesystem added with cytokines such as G-CSF, GM-CSF, and IL-3. Theseresults indicate that MK alone has the activity to maintain theviability of human peripheral blood stem cells or hematopoieticprecursor cells, or promote their proliferation. Furthermore, thecombined use of MK with other cytokines such as M-CSF, G-CSF, GM-CSF,IL-3, and IL-6 synergistically promotes the colony-forming capability.For example, the number of colonies increased 7 to 9-fold in the casesof combined use of MK with G-CSF, GM-CSF, or IL-3 as compared with thoseof the single use of MK, G-CSF, GM-CSF, or IL-3. Also, the combinationof MK+GM-CSF+IL-3+IL-6 remarkably increased the number of coloniesformed as compared with that of GM-CSF+IL-3+IL-6, and the combination ofMK+G-CSF+IL-6 significantly increased the number of colonies as comparedwith that of G-CSF+IL-6. Similarly, when the colony assay was performedwith human peripheral blood mononuclear cells different from those usedin FIG. 1 in the presence of various cytokines and cells constitutingcolonies were morphologically observed as shown in FIG. 2, GM colony wasformed with the single use of MK, GM-CFS, or IL-3, and G colony wasmainly formed with G-CSF alone. Combination of MK+G-CSF+GM-CSF, or thatof MK+G-CSF significantly increased number of G colony as compared withthe single use of G-CSF. That is, MK is considered to synergisticallypromote the proliferation, differentiation and maturation of CFU-GM ofG-CSF, increasing the number of neutrophils in the peripheral blood.

[0019] When cells after 2-week liquid culture of human peripheral bloodstem cells in the presence of various cytokines were examined byspecific staining, there were observed, as shown in FIG. 3,predominantly many granulocytes (neutrophils) in the system supplementedwith MK, clearly indicating the action of MK on the proliferation ofneutrophils. Especially, in the case of combination ofMK+G-CSF+GM-CSF+SCF+IL-3+IL-6, there was observed an extremelyremarkable promotion of the proliferation and differentiation ofneutrophils.

[0020] Also, in the colony assay performed after the above-describedliquid culture, the cell adherence to a culture dish increased in thesystem supplemented with MK as compared with that without MK, indicatingthat MK also promotes the proliferation of interstitial cell system(stroma cell system). In the case of IL-6 alone, colonies formed were ofmacrophages, and in the case of MK+IL-6, half of colonies formed werethose of granulocytes. These results indicate the participation of MK inthe promotion of proliferation and differentiation of granulocytes.

[0021] Whether such a remarkable promotion by MK of the production ofneutrophils is displayed in vivo can be studied by administering MK to amouse whose hematopoietic system has been damaged by administration ofan anticancer drug, or exposure to radiation, and examining the recoverystate of neutrophils. MK was administered to a mouse daily forconsecutive 13 days, and on the 5th day after the initiation ofadministration, an anticancer drug, Cyclophosphamide (CY), wasadministered to the mouse. Examination of hemocytes in the bloodcollected from the mouse at appropriate intervals revealed a remarkablepromotion of the recovery of the number of neutrophils as expected(Table 1). TABLE 1 Leukocyte count (μ/l) Neutrophil count (μ/l) Day 0 24 7 9 Day 0 2 4 7 9 Control 6100 6400 6820 8220 5120 Control 1580 16632046 2606 1204 CY 2000 1040 4020 6560 CY 1330 111 2471 4203 CY + MK 18401100 10460 7640 CY + MK 1077 91 7013 5486 MK 6780 7500 4520 6460 5100 MK1438 2920 1652 2201 2277 Lymphocyte count (μ/l) Erythrocyte (×10⁴/mm³)Day 0 2 4 7 9 Day 0 2 4 7 9 Control 4383 4391 4581 5370 3771 Control 701667 810 832 858 CY 510 866 1220 2034 CY 670 708 687 713 CY + MK 680 9882335 1596 CY + MK 751 679 598 670 MK 4700 3996 2720 3846 2597 MK 818 783792 813 818

[0022] The action of MK on hematopoietic cells under the conditionscloser to in vivo can be examined by, for example, the colony assayusing a methylcellulose medium containing EPO, IL-3, IL-6, and SCF[MethoCult GF M3434 (Stem Cell Technologies, Inc.)]. This medium(hereinafter called M3434 medium) can proliferate and differentiateprecursor cells of erythrocytes, leukocytes, and platelets.

[0023] Results of the colony assay for mouse spleen cells in the systemof the M3434 medium supplemented with MK are shown in FIGS. 4 and 5.FIG. 4 illustrates the number of CFU-GM colony or CFU-G colony. AlthoughCFU-GM colony or CFU-G colony can be formed with the M3434 medium alone,number of colonies generally increases in the system supplemented withthe MK as compared with the M3434 medium alone. Especially, when MK isadded at the concentration of 1 to 10 ng/ml, the colony numberremarkably increased 2 to 3-fold on the 8th and 10th day of the assay.FIG. 5 illustrates the number of colony-forming unit-mixed (CFU-Mix).CFU-Mix are multipotential stem cells at a slightly differentiatedstage, having lower self-renewal capability than blast colony-formingunits (CFU-BL) which are the most undifferentiated cells identifiable bythe in vitro colony assay and are thought to contain cells capable ofdifferentiating to erythrocytes, leukocytes, and platelets. In thesystem supplemented with MK, CFU-Mix colony significantly increased innumber. That is, MK, at least by its combined use with IL-3, IL-6, SCFand EPO, is thought to significantly promote the proliferation anddifferentiation of hematopoietic stem cells or immature hematopoieticcells close to them. These activities are thought to be very useful forthe proliferation of hematopoietic stem cells in vitro for thetransplantation of bone marrow and peripheral blood stem cells, or genetransfer to hematopoietic stem cells.

[0024] In the hematopoietic cells, the more matured peripheral bloodcells are, the more sensitive to anticancer drug. Utilizing thisproperty, the present inventors attempted to concentrate hematopoieticstem cells or hematopoietic precursor cells by an anticancer drug.Namely, the colony assay was performed using spleen cells of a mouse,which had been administered with Cyclophosphamide. Results are shown inFIGS. 6 and 7. FIG. 6 shows the number of CFU-Mix and CFU-G coloniesincreased 2-told or more in the system supplemented with MK as comparedwith the system without MK. This experiment also indicates that MKpromotes the proliferation of hematopoietic stem cells and hematopoieticprecursor cells.

[0025] Effect of MK was investigated using peripheral hemocytes from apatient with non-Hodgkin's lymphoma and a MethoCult H4230 medium[consisting of methylcellulose (0.9%), 2-mercaptoethanol (10 to 4 M),L-glutamine (2 mM), fetal bovine serum (30%), and bovine serum albumin(1%), and containing neither CSF nor EPO; Stem Cell Technologies Inc.]Colony assay was performed using the following combinations; MethoCultH4230 alone, H4230+MK, H4230+G-CSF, and H4230+MK+G-CSF. The ratio ofCD34 positive cells in peripheral stem cells from the patient was 1.4%.Results are shown in FIG. 8. Although no colonies were formed at allwith MK alone, a remarkable colony-forming capability was manifested inthe case of MK+G-CSF, and the number of colonies was clearly twice ormore as high as that in the case of G-CSF alone. On and after the 10thday of the initiation of assay, the increase in number of colonies tendsto reduce in the MK+G-CSF group as compared with the group of G-CSFalone. Microscopic observation of colonies on and after the 10th dayrevealed a tendency that the colony maturation was accelerated in theMK+G-CSF group as compared with the G-CSF alone group. Therefore, thedeceleration of the increase in number of colonies in the MK+G-CSF groupas compared with the G-CSF alone group is probably attributed to theaccelerated maturation of colonies.

[0026] It is noteworthy that, in the above-described colony assay, thesize of each of colonies formed was always larger in groups supplementedwith MK as compared with groups added with no MK. In order to study thisfact quantitatively, three each of large colonies formed on the 14th dayin the presence of MK alone, G-CSF alone, and MK+G-CSF in the colonyassay of peripheral blood stem cells from the above-described patientwere selected, sucked up under a microscope, and counted for theirconstituting cells with a hemocytometer to calculate mean values.Results are shown in FIG. 9. It is obvious that colonies formed withMK+G-CSF contain more constituting cells than those formed with G-CSFalone. That the size of colony is large means that the number ofconstituting cells is also large. From these results, it is evident thatMK acts on the proliferation and differentiation of hematopoietic stemcells and hematopoietic precursor cells.

[0027] The colony assay was similarly performed with the peripheralblood from a healthy normal individual. The ratio of CD34-positive cellsin the peripheral blood of this subject was 0.4%. Results are shown inFIG. 10. Colonies were formed with MK alone. On the 10th and 14th days,the number of colonies increased MK concentration-dependently. TheMK+G-CSG system produced at the highest twice or more as many coloniesas the system of G-CSF alone. These results clearly shows that, when MKwas added, the same tendency was obtained regardless of whether cellsare derived from a healthy normal individual or a patient.

[0028] Furthermore, the colony assay was similarly performed using theperipheral blood from the above-described healthy normal individual inthe presence of pleiotrophin (PTN), which is another member of the MKfamily. PTN used herein was a recombinant PTN [pleiotrophin, recombinanthuman (Sf21-derived); Lot GH055011) (R & D Systems)]. Results are shownin FIG. 11. PTN alone exhibited the colony-forming capability like MKand the number of colonies formed was markedly high. A synergisticaction of PTN with G-CSF to promote the colony formation was similarlyobserved as in the case of MK. From these results, PTN obviouslypromotes the proliferation and differentiation of hematopoietic stemcells and hematopoietic precursor cells like MK.

[0029] Next, using a hematopoietic stem cell assay medium, complete type(Lot No. 96101601; Kyokuto Seiyaku Kogyo) containing IL-3, SCF, G-CSF,and EPO, the colony assay was carried out with peripheral blood cellsfrom a healthy normal individual. This assay is considered to beperformed under conditions closer to in vivo. Results with MK are shownin FIGS. 12 and 13, and those with PTN in FIGS. 14 and 15. No increasein BFU-E was observed with any combinations including MK+IL-3, MK+SCF,and MK+G-CSF. However, the combinations of MK+EPO and PTN+EPO wereassumed to increase BFU-E. Erythroblast precursor cells, BFU-E, wereformed on the 14th day of culture, and are known to be moreundifferentiated than CFU-E formed on the 5th to 7th days. Addition ofMK or PTN to a Kyokuto complete medium resulted in formation of at thehighest twice or more as many BFU-E as the complete medium alone on the12th day after the initiation of culture. These results indicate that atleast the addition of MK to the complete medium results in promoting theproliferation of erythroblasts as well.

[0030] As described above, it is evident that the MK family is capableof acting on hematopoietic stem cells and hematopoietic precursor cellsin the hematopoietic tissues of mammals to maintain, proliferate, anddifferentiate them, and synergistically or additionally enhancing theabove-described functions by the combined use with various cytokinessuch as SCF, M-CSF, G-CSF, GM-CSF, IL-3 and IL-6. Especially, the MKfamily remarkably promotes the proliferation of CFU-Mix, which is veryclose to multipotential stem cells, under conditions closer to in vivo.The MK family also promotes the proliferation and differentiation ofgranulocyte/macrophage precursor cells and exerts the remarkableneutrophil increasing effect in an in vivo neutropenia model. This MKfamily alone or in combination with more than one kinds of cytokinesincluding SCF, M-CSF, G-CSF, GM-CSF, IL-3, and IL-6 can be clinicallyapplied and, especially, used for the ex vivo expansion of hematopoieticstem cells in the transplantation of bone marrow and stem cells derivedfrom the peripheral blood and umbilical cord blood. In addition, the MKfamily is expected to be used for the treatment and prevention ofpatient with neutropenia, vertebrate anemia, and leukemia caused bycancer chemotherapy. Furthermore, the MK family would be used forproliferating stem cells for gene therapy targeting hematopoietic stemcells in future. Especially, it is very promising to increase the dosedensity in cancer chemotherapy by the combined use of MK with G-CSF,improving effects of chemotherapy by increasing the dose of antitumordrugs or shortening the administration period.

[0031] MK and PTN used in the present invention can be either a naturalor recombinant product. The recombinant MK family means a substance wellhomologous with the natural MK or PTN and having biological activitiesequivalent thereto. Such MK or PTN includes their derivatives andanalogues. The purified MK or PTN of mammals means those derived frommice or humans, but not limited thereto. MK or PTN of this inventionalso includes glycosylated and non-glycosylated MK or PTN.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 illustrates effects of the single or combined use of MK,G-CSF, GM-CSF, M-CSF, IL-3, and IL-6 on the colony-forming capability ofhuman peripheral blood mononuclear cells.

[0033]FIG. 2 illustrates effects of the single or combined use of MK,G-CSF, GM-CSF, M-CSF, IL-3, IL-6 and SCF on G colony, GM colony and Mcolony-forming capabilities of mononuclear cells in the human peripheralblood different from that used in the experiment in FIG. 1.

[0034]FIG. 3 illustrates numbers of granulocytes, monocytes, ormacrophages, and other cells counted by the esterase double staining ofhuman peripheral blood mononuclear cells after two-week liquid-culturein the presence of MK, G-CSF, GM-CSF, IL-3, IL-6, and SCF alone or incombination.

[0035]FIG. 4 illustrates effects of MK on the colony-forming capabilityof mouse spleen cells cultured in a complete methylcellulose mediumcontaining EPO, IL-3, IL-6, and SCF (MethoCult GF M3434) supplementedwith MK for 12 days in order to examine the proliferation promotingaction of MK on hematopoietic cells under conditions closer to in vivo.

[0036]FIG. 5 illustrates effects of MK on the CFU-Mix colony-formingcapability in an experiment similar to that in FIG. 4.

[0037]FIG. 6 illustrates effects of MK on the CFU-G colony-formingcapability when an anticancer drug, Cyclophosphamide, was administeredto a mouse, myelocytes were isolated on the 4th day after the drugadministration and cultured in a complete methylcellulose mediumcontaining EPO, IL-3, IL-6, and SCF (MethoCult GF M3434) supplementedwith MK for 14 days.

[0038]FIG. 7 illustrates effects of MK on the CFU-Mix colony-formingcapability in an experiment similar to that in FIG. 6.

[0039]FIG. 8 illustrates effects of MK on the colony-forming capabilitywhen the peripheral blood from a patient with non-Hodgkin's lymphomawere cultured in a methylcellulose medium for the colony assay(MethoCult GF H4230) supplemented with MK, G-CSF, or MK+G-CSF for 14days.

[0040]FIG. 9 illustrates the number of colony-constituting cells on the14th day in an experiment similar to that in FIG. 8 where the mediumalone and that supplemented with G-CSF or MK+G-CSF was used.

[0041]FIG. 10 illustrates effects of MK and G-CSF on the colony-formingcapability when the peripheral blood from a healthy normal individualwas cultured in a methylcellulose medium for the colony assay (MethoCultGF H4230) supplemented with MK, G-CSF, or MK+G-CSF.

[0042]FIG. 11 illustrates results of an experiment similar to that inFIG. 10 where PTN was used in place of MK.

[0043]FIG. 12 illustrates effects of MK on the CFU-E colony-formingcapability when the same human peripheral blood used in the experimentof FIG. 10 was cultured in a medium for the blood stem cell assaycontaining EPO, IL-3, G-CSF, and SCF supplemented with MK.

[0044]FIG. 13 illustrates effects of MK on the colony-forming capabilityof BFU-E, which is more undifferentiated than CFU-E, in an experimentsimilar to that in FIG. 12.

[0045]FIG. 14 illustrates effects of PTN on the CFU-E colony-formingcapability when the peripheral blood from the same healthy normalindividual used in the experiment of FIG. 10 was cultured in the samemedium used in the experiment of FIG. 10 supplemented with PTN in placeof MK.

[0046]FIG. 15 illustrates effects of PTN on the BFU-E colony-formingcapability in an experiment similar to that in FIG. 14.

BEST MODE FOR IMPLEMENTING THE INVENTION

[0047] The present invention will be described below with reference toexamples, but is not to be construed to be limited thereto. MK usedherein is human MK described in SEQ ID NO: 3 in Japanese PatentApplication No. Hei 7-255354.

EXAMPLE 1 Effect of MK on Promoting Neutrophil Recovery in a NeurtopeniaModel

[0048] Neutropenia is a disease wherein neutrophils that play the mostimportant role in preventing infection are selectively lost orsignificantly reduced in number. In the following is presented anexample, wherein MK was administered to a neutropenia model prepared byadministering an antitumor drug to normal mice and examined for itseffect on promoting neutrophil recovery.

[0049] Neutropenia model mice were prepared by administering anantitumor drug, Cyclophosphamide (CY) to 12-week-old ICR mice (male).The mice were divided into the following groups so that each group hadfive mice; (1) untreated group (control group), (2) CY-administeredgroup, (3) CY+MK-administered group, and (4) MK alone administeredgroup. MK was diluted with a physiological saline and intrapenitoneallyadministered to the mice daily at a dose of 0.1 ml/animal and 300 μg/kgfor 13 consecutive days. Six hours later the administration on the 5thday of the consecutive administration, CY was administered to the miceat a dose of 266 mg/kg, corresponding to ⅔ of the LD₅₀ value. The day ofCY administration was taken as Day 0, and the blood was collected fivetimes in total, namely on Day 0, Day 2, Day 4, Day 7, and Day 9, andcounted for leukocytes, neutrophils, lymphocytes, and erythrocytes.Results are shown in Table 1. The number of lymphocytes reached thelowest value on Day 2 in the CY-administered group and theCY+MK-administered group as compared with the control group, and did notrecover until Day 9. The number of neutrophils reached the lowest valueon Day 4, but elevated to 2.8 times as high as that of the control groupon Day 7.

EXAMPLE 2 Colony Formation Promoting Action of MK and other Cytokines onHuman Peripheral Blood Mononuclear Cells

[0050] For collecting human peripheral blood stem cells (PBSC), it isnecessary to let them migrate from the bone marrow to the peripheralblood. A hematopoietic factor, G-CSF, was administered to an individual,who is in the hematologically stable conditions, to induce migration ofPBSC to the peripheral blood, and the blood was collected with aheparinized syringe. The peripheral blood was fractionated using aseparation agent. The mononuclear cell layer thus fractionated was mixedwith a phosphate buffer (PBS), and centrifuged at 4° C. and 1500 rpm for5 min. After the centrifugation, the supernatant was discarded and cellswere washed by repeating this procedure several times. The cells weresuspended in a medium containing 10% FBS and counted with hemocytometerK-8000. Finally, the cell concentration was adjusted to 1×10⁶/ml with amedium containing 10% FBS.

[0051] Test substances used were MK, 50 μg/ml; G-CSF, 10 ng/ml; GM-CSF,10 ng/ml; M-CSF, 50 ng/ml; IL-3, 10 ng/ml; IL-6, 100 ng/ml; and SCF, 10ng/ml. These substances were adjusted to have the above concentrationsby preparing each solution with 10-fold concentration of the finalconcentration using Iscove's Modified Dulbecco's Medium (IMDM) andadding it to an assay system in an amount of 10% of the total volume ofthe system.

[0052] A methylcellulose medium containing FBS was used for the colonyassay. A methylcellulose solution was prepared by adding to the mediummethylcellulose powder (3500 to 5600 cps, Tokyo Kasei Kogyo) to 2%.

[0053] A necessary amount of each solution prepared as described abovewas placed in a single tube. Namely, in each tube, solutions were mixedto give final concentrations of 1×10⁵/ml for cells, 20% for FBS, 20% forthe medium containing 10% FBS, and 0.8% for methylcellulose. The testsubstances were then respectively added to this mixture to give theabove-described concentrations. After the resulting mixture wasthoroughly vortexed, it was inoculated onto a plastic culture dish(Falcon, 1008) using a syringe with a #18 needle and cultured in a 5%carbon dioxide incubator at 100% humidity and 37° C. for 2 weeks. Aftertwo weeks, the number of colonies formed was counted using an invertedmicroscope. Results are shown in FIG. 1.

[0054] The colony assay of peripheral blood mononuclear cells fromanother healthy normal individual using G-CSF, GM-CSF, and M-CSF wasperformed by a similar method as described above. Results are shown inFIG. 2.

EXAMPLE 3 Liquid Culture of Human Peripheral Blood Mononuclear Cells inthe Presence of MK and other Cytokines

[0055] Human peripheral blood mononuclear cells and test substances wereprepared similarly as in Example 2. The test substances wererespectively added to the mixture containing 1.5×10⁵/ml of cells, 30% ofFBS, and 10% of the medium containing 10% BSA to give theabove-described concentrations. The resulting mixture was distributedonto a plastic culture dish (Falcon, 1008) and cultured in a 5% carbondioxide incubator at 100% humidity and 37° C. for 2 weeks.

[0056] After 2 weeks, all cells were recovered from each culture dish.Cells adhering to the culture dish were recovered by treating them with0.25% trypsin/EDTA. Cells were recovered in tubes and centrifuged onceat 4° C. and 800 rpm. The thus-collected cells were suspended in thesame volume of the culture medium and counted with a hemocytometer.

[0057] The cells collected for counting were mounted onto a slide glasswith Cytospin (Cytospin 2; SHANDON) and subjected to the esterase doublestaining (an esterase staining kit and esterase AS-D staining kit, MutoKagaku Yakuhin). Changing the field of vision, cells were distinguishedinto granulocytes, monocytes, macrophages, and others according to thestainablity. Results are shown in FIG. 3.

EXAMPLE 4 Hematopoiesis Promoting Action of MK Under Conditions Closerto in Vivo

[0058] The spleen of 8-week-old BDF1 mouse (female) was asepticallyexcised in a clean bench, and cells were pressed out using a needle[Therumo,22G ×1¼″ (0.70×32 mm)] into IMDM (GIBCO BRL) in a petri dish.Cells in the spleen were collected into IMDM (10 ml) in a tube,thoroughly mixed by pipetting, and passed through a cell strainer(FALCON 2350, 70 μm). Mononuclear cells were counted with ahemocytometer, adjusted to the concentration 1×10⁶ cells/ml with IMDM toserve as a cell suspension. Test solutions containing MK at 100 ng/ml, 1mg/ml, and 10 mg/ml in IMDM were similarly prepared.

[0059] The methylcellulose medium used was a complete medium, MethoCultGF M3434 (containing 0.9% methylcellulose, 10 to 4 M 2-mercaptoethanol,2 mM L-glutamine, FBS, 1% BSA, EPO, insulin, transferrin, IL-3, IL-6 andSCF; Stem Cell Technologies, Inc.). To the medium were added theabove-described cells to 1×10⁵/ml and MK to a final concentration of 1,10, or 100 ng/ml. The colony assay was then performed by a similarmethod as in Example 2. Results are shown in FIGS. 4 and 5.

EXAMPLE 5 Hematopoiesis Promoting Action of MK under Conditions Closerto in Vivo (Concentration of Hematopoietic Stem Cells and HematopoieticPrecursor Cells)

[0060] To concentrate hematopoietic precursor cells in myelocytes,Endoxan powder [powerful drug according to Japanese Pharmacopoeia,Cyclophosphamide (CY)] was administered to five 8-week-old BDF1 mice(female) at a dose of 1 mg/animal. Four days after the administration,cells were prepared from the mouse bone marrow by the usual method, andadjusted to the concentration of 2×10⁴ cells/ml with IMDM. The colonyassay was then performed in the same manner as in Example 4. Results areshown in FIGS. 6 and 7.

EXAMPLE 6 Effects of MK and Other Cytokines on the Colony Formation ofPeripheral Blood Mononuclear Cells from a Patient with Non-Hodgkin'sLymphoma

[0061] The medium used was a methylcellulose medium, MethoCult H4230(containing a final concentration of 0.9% methylcellulose, 10 to 4 M2-mercaptoethanol, 2 mM L-glutamine, 30% FBS, and 1% bovine serumalbumin; Stem Cell Technologies, Inc.). Results are shown in FIGS. 8 and9.

EXAMPLE 7 Effects of MK and PTN on the Colony Formation of PeripheralBlood Mononuclear Cells from Healthy Normal Individual

[0062] The colony assay was carried out using the same medium used inExample 6. Results are shown in FIGS. 10 and 11.

EXAMPLE 8 Effects of MK and PTN on the Erythroblast Colony Formation ofPeripheral Blood Mononuclear Cells from Healthy Normal Individual

[0063] The colony assay was carried out using a hematopoietic stem cellassay medium (complete type, Kyokuto Seiyaku Kogyo, containing 30% FCS,1% BSA, 10 to 4 M 2-mercaptoethanol, IMDM, PS solution, 1.2%methylcellulose, 10 ng/ml IL-3, 10 ng/ml G-CSF, 2 U/ml EPO, and 50 ng/mlSCF). Results are shown in FIGS. 12, 13, 14, and 15.

[0064] Industrial Applicability

[0065] The MK family acts on hematopoietic stem cells and precursorcells of various hemocytes of hematopoietic tissues of mammals tomaintain, proliferate, and differentiate them. Furthermore, theabove-described functions are synergistically or additionally enhancedby the combined use or MK with other cytokines such as SCF, M-CSF,G-CSF, GM-CSF, IL-3, and IL-6. Especially, the MK family exertsremarkable proliferation promoting effects on CFU-Mix, which is veryclose to multipotential stem cells, under conditions closer to in vivo.The MK family also promotes the proliferation and differentiation ofprecursor cells of the granulocytes/macrophages and remarkably increaseneutrophils in neutropenia in vivo. The pharmaceutical composition ofthe present invention containing the MK family alone, or containing theMK family in combination with one or more cytokines such as SCF, M-CSF,G-CSF, GM-CSF, IL-3, and IL-6 can be clinically applied, especially, tothe ex vivo expansion of stem cells in the transplantation of bonemarrow and stem cells derived from the peripheral blood and umbilicalcord blood. The MK family is also expected to be used for treating andpreventing neutropenia, inveterate anemia, and leukemia caused by thecancer chemotherapy. Furthermore, the MK family is expected to be usedfor the stem cell proliferation for gene therapy targeting hematopoieticstem cells.

1. An agent for promoting proliferation and differentiation ofhematopoietic stem cells and/or hematopoietic precursor stem cells ofmammals comprising a protein belonging to the MK family as an activeingredient.
 2. The agent of claim 1, which comprises a protein belongingto the MK family and one or more other hematopoietic factors as activeingredients.
 3. The agent of claim 2, wherein said other hematopoieticfactors are selected from a group consisting of IL-3, IL-6, G-CSF,GM-CSF, M-CSF, SCF, and EPO.
 4. The agent of claim 3, wherein said otherhematopoietic factors are IL-3, IL-6, SCF, and EPO.
 5. The agent ofclaim 3, wherein said other hematopoietic factors are IL-3, IL-6, G-CSF,GM-CSF, and SCF.
 6. The agent of claim 3, wherein said otherhematopoietic factor is G-CSF.
 7. The agent of any one of claims 1 to 6,wherein said hematopoietic stem cells are CFU-Mix.
 8. Use of a proteinbelonging to the MK family for preparing an agent for promotingproliferation and differentiation of hematopoietic stem cells and/orhematopoietic precursor cells of mammals.
 9. A method for promotingproliferation and differentiation of hematopoietic stem cells and/orhematopoietic precursor cells of mammals, comprising administering aprotein belonging to the MK family.
 10. Use of a protein belonging tothe MK family and one or more other hematopoietic factors for preparingan agent for promoting proliferation and differentiation ofhematopoietic stem cells and/or hematopoietic precursor cells ofmammals.
 11. A method for promoting proliferation and differentiation ofhematopoietic stem cells and/or hematopoietic precursor cells ofmammals, comprising administering a protein belonging to the MK familyand one or more other hematopoietic factors.
 12. A pharmaceuticalcomposition for treating neutropenia of mammals, comprising a proteinbelonging to the MK family and one or more other hematopoietic factorsas active ingredients.
 13. The pharmaceutical composition of claim 12,wherein said other hematopoietic factor is G-CSF.
 14. Use of a proteinbelonging to the MK family and one or more other hematopoietic factorsfor preparing a pharmaceutical composition for treating neutropenia ofmammals.
 15. A method for treating neutropenia of mammals, comprisingadministering a protein belonging to the MK family and one or more otherhematopoietic factors.
 16. A pharmaceutical composition for ex vivoexpansion of hematopoietic stem cells for the transplantation of bonemarrow and peripheral blood stem cells, comprising a protein belongingto the MK family and one or more other hematopoietic factors as activeingredients.
 17. The pharmaceutical composition of claim 16, whereinsaid other hematopoietic factors are selected from a group consisting ofIL-3, IL-6, G-CSF, GM-CSF, M-CSF, SCF, and EPO.
 18. The pharmaceuticalcomposition of claim 17, wherein said other hematopoietic factors areIL-3, IL-6, SCF, and EPO.
 19. Use of a protein belonging to the MKfamily and one or more other hematopoietic factors for preparing apharmaceutical composition for ex vivo expansion of hematopoietic stemcells for the transplantation of bone marrow and peripheral blood stemcells.
 20. A method for ex vivo expansion of hematopoietic stem cellsfor the transplantation of bone marrow and peripheral blood stem cells,comprising administering a protein of the MK family and one or moreother hematopoietic factors.